ABSTRACT The mammalian brain is composed of 50% neurons and 50% non-neuronal glial cells, including astrocytes (20%) which regulate synaptic transmission and provide metabolic support for neurons, oligodendrocytes (25%) that speed up neuronal action potential conduction, and microglia (5-15%) which are tissue-resident macrophages with important roles in homeostasis and protection from infection. The ability to genetically target subsets of glial cells within a circuit, e.g. astrocytes in a given layer of the cortex, or astrocytes in the cortex and not in the thalamus, is lacking. Further, the ability to specifically target glial cells at early stages of development, without targeting other cells including neurons, is a challenge due to the promoters used often being expressed embryonically by radial glia that give rise to both neurons and glial cells. Therefore, there is a pressing need to identify new genetic points of entry to specific subsets of glia within a circuit at specific times, in order to allow cell-type specific manipulation to interrogate the role of glia in circuit function. To achieve this, a systematic classification of glial cell types and subtypes in the adult and developing brain is required. This project will approach this within the visual system of the mouse, using an innovative, recently developed single- cell multi-omics method to profile transcriptome and methylome from the same cell, called single-nucleus methylCytosine and Transcriptome sequencing (snmCT-seq). Applying snmCT-seq to study glial cell diversity will lead to a high-resolution classification of glial cell types across developmental stages and major regions of the mouse brain. Aim 1 will use snmCT-seq to profile glial cells from the adult (4 month) mouse visual system - retina (input region), dLGN (thalamic relay) and visual cortex (target area). This will identify cell-type and region-type specific regulatory elements that can be used to genetically access glial cells. In Aim 2 this project will analyze the same brain regions as a timecourse across late embryonic and early postnatal development, to identify stage-specific regulatory elements that can be used to target glial cells in early development. Aim 3 will test candidate regulatory elements for the ability to drive transgene expression in specific sub-sets of glial cells at specific developmental timepoints, using viral delivery strategies, to generate new genetic access to glial cell types. The outcomes of this work will provide an inventory of glial sub-types within a defined circuit, identify stage- and region-specific markers of glial cells, and identify regulatory elements that can target subtypes of glia. The viral tools developed in Aim 3 will enable researchers to target and manipulate glial cells in a circuit- specific manner, to interrogate the contribution of glial cells to neuronal circuit function. The use of viruses will allow use in multiple mammalian species, expanding the utility of this resource.